Microbubble and nanobubble expansion using perfluorocarbon nanodroplets for enhanced ultrasound imaging and therapy

ABSTRACT

The disclosure describes imaging and therapy techniques comprising nanodroplets. More particularly, aspects of the disclosure relate to the use of nanodroplets to modify nanobubbles or microbubbles to provide improved imaging and/or therapeutic techniques and compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 62/809,280 filed Feb. 22, 2019, the entire contents of whichare incorporated herein by reference, including the appendices attachedthereto.

BACKGROUND

The invention was made with government support under and CancerPrevention Research Institute of Texas (CPRIT) RR150010, DOD Idea Award14W81XWH-17-1-0401 and HCC Development Project Pilot Award (Internal—preSPORE). The government has certain rights in the invention.

A. Field

This disclosure relates to imaging and therapy techniques comprisingnanodroplets. More particularly, embodiments of the disclosure relate tothe use of nanodroplets to inflate nanobubbles or microbubbles toprovide improved imaging and/or therapeutic aspects.

B. Related Art

Various techniques have been developed to image and provide therapy toconditions that require medical treatment, including for example,cancerous tumors. Certain techniques incorporate the use of gaseousbubbles to increase the visibility of regions of interest duringultrasound imaging. Acoustic droplet vaporization (ADV) requiresacoustic waves to decrease local pressure of liquid perfluorocarbon(PFC) below its vapor pressure to trigger its phase transition fromliquid to gas. Although promising, this field remained stagnant despitethe introduction of novel formulations of low-boiling point (<0° C.)perfluorocarbon (PFC) nanodroplets (NDs) that are superheated at bodytemperature. This is most likely due to the larger ND dose neededcompared to microbubbles (MBs), the inability to vaporize all NDs, theamount of phospholipid surfactant that is insufficient to properlystabilize newly formed MBs that are five times larger, and thespontaneous uncontrolled vaporization of ND that could lead to potentialtoxicity.

Neat perfluorocarbon (PFC) and emulsions of PFC exhibit exceptionalproperties such as high thermal stability and chemical inertness owingto the unique characteristics of fluorine (F) and the carbon-fluorine(C—F) linkage—the strongest covalent bond in organic chemistry. PFC inits liquid state is often considered gas-like fluid as it displays lowintermolecular cohesiveness due to the low polarizability of F, whichresults in lower Van der Waals (VdW) interactions between pairs of CF₂as opposed to VdW interactions between pairs of CH₂. More importantly,PFC have additional interrelated key properties that are critical forclinical translation, including biocompatibility, high purity, lowsurface tensions, high fluidity as well as high vapor pressure and gassolubility compared to hydrocarbons of similar molecular mass (1). Inaddition, low molecular weight PFC are rapidly excreted by exhalationfor PFC with no side effects.

Owing to their unique ultrasound (US) backscattered signal, single PFCgas-filled microbubbles (MBs) (2, 3) or single MB-labeled cells (4) canbe detected in vivo using a clinical US system. This unique US responseof PFC gas-filled MBs has prompted the development of a variety of newUS contrast agents and has increased the possibility of diagnosticimaging worldwide. However, other than altering ligands on the MBshellto target specific tissues, translatable MB-based discoveries as UScontrast agents have been stagnant for the past 2 decades, mostlybecause their micron size limits their distribution to the intravascularspace where they can only interact with the endothelial surface (5, 6),circulating cells (7), or blood clots (8, 9).

Nanoscale particles exhibit several advantages for molecular imagingover MBs. Because of their 100-300 nm size, PFC nanodroplets (NDs) areable to leak into the tumor extracellular space (1). In addition, theirlarger particle count, greater resilience to US and longer in vivo dwelltime are expected to improve targeting efficiency of NDs over MBs tointravascular sites in addition to extravascular sites. However, the keyimpediment of NDs is their lesser echogenicity and thus lesser UScontrast compared to MBs due to their limited reflectivity andcompressibility (10), requiring large doses that potentially increaseside-effects (11). While their backscatter is indistinguishable fromtissues, NDs are able to phase change into MBs through a process knownas of acoustic droplet vaporization (ADV) resulting in improveddetection by US. ADV was introduced almost two decades ago as apotential tool to induce vascular occlusion by locally triggering localembolization on downstream tissues using micron-size perfluoropentane(PFP) filled droplets (12). However, using micron-size PFP-filleddroplets potentially leads to spontaneous droplet vaporization,particularly during injection due to shear stress and cavitation, whichis a concern for clinical translation (13). In addition, occlusion wasaccomplished exclusively in the larger arteries, resulting in undesirednon-discriminant downstream embolization and side effects (14-16) orincomplete embolization for tumors still fed from smaller arteries.

While a few groups have successfully vaporized liquid low boiling pointdroplets of PFP (17), or perfluorobutane (PFB) (18-20) to MBs inanimals, their clinical translation and widespread use has been limitedby critical efficacy and safety challenges. Firstly, the acoustic energyrequired to vaporize droplets, particularly NDs, in humans is expectedto be higher than the current FDA limit (12) because of the high Laplacepressure inside NDs, the increased interstitial pressure in tumors andthe US attenuation in deep tissues (21). Secondly, the required dose ofdroplets or NDs is high, which potentially leads to side-effects (11).Thirdly, the inability to vaporize all NDs due to tissue attenuation,and uncontrolled spontaneous vaporization of sub-micron droplets at bodytemperature (13) raises safety concerns as it could lead to unwantedmicrovascular occlusion. Finally, red blood cell extravasation occursfollowing ADV-triggered capillary rupture due to initial MB expansion ormost likely to the violent MB collapse associated with inertialcavitation at insonation (22). In addition to these safety concerns, MBsformed by ADV might not be stable enough for effective gas embolotherapyas they originate from NDs that are 5 times smaller (23), and thus donot have enough phospholipid surfactants to be properly stabilized.Although the presence of MBs near NDs decreases the ADV threshold (24),the same challenges remain for the clinical translation of ADV.

SUMMARY

Embodiments of the present disclosure relate to the field of medicaldiagnostics and therapeutics, and more specifically to the developmentof a new class of imaging and theranostic agents obtained by thecombination of perfluorocarbon (PFC) gas filled microbubbles (MBs) andnanobubbles (NBs) with PFC liquid core nanodroplets (NDs). Particularaspects present a novel strategy for improved detection and treatment ofdiseased tissue. Certain aspects comprise using ultrasound contrastagents (MBs or NBs) to target intravascular and/or extravascular sites,followed by a second injection of non-targeted NDs or NDs targeted tothe pre-injected MBs or NBs. In specific aspects, the PFC liquid pool ofNDs may then transfer to the MBs or NBs to inflate them. Inflating MBsor NBs may increase ultrasound signal easing detection. However,inflation may be used to speed blood clot dissolution, induce tumorvascular occlusion, aid in gene delivery and transfection, and more.

The inventors have found that NDs act as a PFC and phospholipidreservoirs that transfer to the adjacent NBs or MBs, which triggerstheir growth. Interestingly, while both NBs and NDs individually exhibitvery limited ultrasound signal on B-mode imaging when exposed to lowacoustic power, their combination results in a rapid and dramaticincrease in signal visible on standard ultrasound imaging. The inventorshypothesize that the low but non-negligible PFC solubility in water andhigh vapor pressure of PFC allow condensed PFC from the NDs to dissipateand diffuse away into the aqueous phase to reach the NB core andinitiate expansion. This innovation is different from the use of NDs asphase change contrast agent (PCCA) because no sound waves are necessaryto inflate the NBs or MBs but result from the large vapor pressure inthe superheated nanodroplets. Such a driving force does not existbetween two microbubbles with the same Laplace pressure.

The inventors have demonstrated rapid NB expansion when mixed with aconcentrated solution of fluorescein-labeled NDs (˜10:1 ND/NB ratio) bymicroscopy and ⋅ultrasound. The inventors showed evidence that thephospholipids from the ND shell were incorporated with the newly formedmicrobubbles (MBs) by fluorescence microscopy.

Embodiments of the present disclosure also relate to the field of invitro rare cell isolation. More specific aspects describe a new platformthat may improve cell detection sensitivity and isolation using acombination of perfluorocarbon (PFC) gas filled nanobubbles (NBs) ormicrobubbles (MBs) targeted to any cell surface receptor of interest,and PFC liquid nanodroplets (NDs). Particular aspects include a novelstrategy to better detect and isolate cells in vitro using buoyancy.Certain aspects comprise adding targeted NBs or MBs to a cellsuspension, followed by the addition of NDs that could be non-targetedor targeted to the MBs or MBs. The liquid PFC from superheated PFC NDscan then transfer to the NBs or MBs to inflate them, increase buoyancyand the floatation of MB-cell construct to ease or speed their recoveryusing mild or no centrifugation.

Briefly, the present disclosure provides a novel strategy to achieve thebenefits of ADV, including improved detection and tumor microvascularembolization, without the need for ultrasound. Because liquid PFC in NDsis at a much higher partial pressure than its gaseous counterpart inmicrobubbles (MB), the coexistence of ND and MB leads to PFCvaporization and transfer into MB leading to their expansion. Thethermodynamic driving force is the large vapor pressure for thenanodroplets, which are highly superheated. Such a driving force doesnot exist between two microbubbles with the same Laplace pressure. Theextent of the MB expansion was quantified by counting and measuring thesize of expanded microbubbles with microscopy and flow cytometry. ThisMB expansion process was applied to expand poorly echogenic nanobubbles(NBs) to larger MBs and resulted in a 4-fold ultrasound signalenhancement after 30 seconds of incubation at 37° C. The inventors havedemonstrated that the rate and extent of expansion were affected by thetype of PFC used, MBsize, length and type of lipid emulsifier that hasan effect on both interfacial tension and the ability of bubbles toexpand. Finally, MB inflation was demonstrated in vitro in tubing flowsystem and in two animal models. First, MBs and ND were both injectedsubcutaneously in the ear of a mouse to demonstrate MB expansion in vivousing intravital microscopy. In a second animal model, MC38 colon cancercells were implanted in the flank of a mouse and MC38-targeting MBs andNDs were injected intravenously in sequence to demonstrate tumortargeting and MB inflation at the tumor site using microscopy.

Certain embodiments include a method of imaging a region of interest,where the method comprises: providing bubbles to the region of interest;providing nanodroplets to the region of interest; increasing the averagebubble diameter of the bubbles while the bubbles are in the region ofinterest; and exposing the region of interest to an ultrasound stimulusto generate an image of the region of interest.

Certain embodiments include a method to safely occlude tumormicrovessels non-invasively and without the need for ultrasound, wherethe method comprises: providing bubbles to the region of interest,diameter between 200 nm and 10 μm; providing nanodroplets to the regionof interest; increasing the average bubble diameter of the bubbles whilethe bubbles are in the region of interest.

In particular embodiments, providing the nanodroplets to the region ofinterest is performed subsequent to providing the bubbles to the regionof interest. In some embodiments, the bubbles and the nanodroplets areprovided to the region of interest in a composition comprising thebubbles and the nanodroplets. In specific embodiments, the averagebubble diameter of the bubbles is increased by an order of magnitudewhen the bubbles are in the region of interest. In certain embodiments,the average bubble diameter of the bubbles is increased by up to twoorders of magnitude when the bubbles are in the region of interest. Inparticular embodiments, the nanodroplets comprise a perfluorocarbon(PFC).

Specific embodiments include a method of increasing bubble size, wherethe method comprises: providing bubbles to a first region, wherein thebubbles have an average bubble diameter between 200 nm and 10 μm;providing nanodroplets proximal to the bubbles; and increasing theaverage bubble diameter of the bubbles while the bubbles are in thefirst region. In certain embodiments, providing the nanodropletsproximal to the bubbles is performed subsequent to providing the bubblesto the first region. In particular embodiments, the bubbles and thenanodroplets are provided in a composition comprising the bubbles andthe nanodroplets. In some embodiments, the average bubble diameter ofthe bubbles is increased by an order of magnitude when the bubbles arein the region of interest.

Particular embodiments include a method of isolating a cell populationin blood or media, where the method comprises: providing bubbles to aregion of interest, wherein the bubbles are coupled to targeted cellstructures within the region of interest; providing nanodroplets to theregion of interest; increasing the average bubble diameter of thebubbles and increasing the buoyancy of the bubbles while the bubbles arecoupled to the targeted cell structures within the region of interest;and isolating the bubbles and targeted cell structures within the regionof interest from non-targeted cell structures within the region ofinterest.

In certain embodiments, isolating the bubbles and targeted cellstructures within the region of interest from non-targeted cellstructures within the region of interest comprises floating the bubblesand targeted cell structures within the region of interest. Inparticular embodiments, providing the nanodroplets to the region ofinterest is performed subsequent to providing the bubbles to the regionof interest. In some embodiments, the bubbles and the nanodroplets areprovided to the region of interest in a composition comprising thebubbles and the nanodroplets. In specific embodiments, the averagebubble diameter of the bubbles in increased by an order of magnitudewhen the bubbles are in the region of interest. In certain embodiments,the nanodroplets comprise a perfluorocarbon (PFC).

Any embodiment of any of the present methods, composition, kit, andsystems may consist of or consist essentially of—rather thancomprise/include/contain/have—the described steps and/or features. Thus,in any of the claims, the term “consisting of” or “consistingessentially of” may be substituted for any of the open-ended linkingverbs recited above, in order to change the scope of a given claim fromwhat it would otherwise be using the open-ended linking verb.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

Following long-standing patent law, the words “a” and “an,” when used inconjunction with the word “comprising” in the claims or specification,denotes one or more, unless specifically noted.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating certain embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein. The patent or application filecontains at least one drawing executed in color. Copies of this patentor patent application publication with color drawing(s) will be providedby the Office upon request and payment of the necessary fee.

FIG. 1 illustrates a flowchart of steps in a method that can be used inultrasound imaging techniques according to an exemplary embodiment ofthe present disclosure.

FIG. 2 illustrates a flowchart of steps in a method for providing atherapeutic effect to a region of interest.

FIG. 3 illustrates a flowchart of steps in a method that combinesaspects of imaging method and treatment.

FIG. 4 illustrates a flowchart of steps in a method for increasing anaverage bubble diameter, without specification to imaging or therapeuticaspects.

FIG. 5 illustrates a flowchart of steps in a method for using ex vivonanodroplets to increase the average bubble diameter and buoyancy of themicrobubble-cell complexes in a blood sample

FIG. 6 illustrates representative bright field images of NDs and NBsprior and after mixing at 20× magnification.

FIG. 7 illustrates representative bright field and fluorescence imagesof NDs before and after mixing with NBs at 20× magnification.

FIG. 8 illustrates a schematic representation of a one-compartmentexperimental setup.

FIG. 9 illustrates shows a schematic representation of a two-compartmentexperimental setup.

FIG. 10 illustrates a schematic representation of thenanodroplet-microbubble gas and phospholipid exchange.

FIG. 11 illustrates PFB nanobubbles inflation triggered by the additionof PFB nanodroplets.

FIG. 12 illustrates an experimental setup and bright-field microscopyimages showing a large amount of expanded MBs.

FIG. 13 illustrates an optically transparent two-compartment systemseparated by a semi-permeable membrane between a microscopy slide and acoverslip to observe MB inflation in real time using bright-fieldmicroscopy.

FIG. 14 Sections A and B illustrate that both lipophilic payload andphospholipid shell transfer during MB inflation, using microscopy andflow cytometry. Section C illustrates an experimental setup equivalentto that of FIG. 13 using fluorescently labeled MBs and NDs.

FIG. 15 illustrates the different inflation kinetics between albumin-MBsand phospholipid based MBs.

FIG. 16 includes bright-field microscopy and graphical images showingthe effect of ND PFC core on PFB MBs inflation.

FIG. 17 includes bright-field microscopy and graphical images showingthe effect of bubble size on inflation.

FIG. 18 includes bright-field microscopy and graphical images showingthe effect of bubble size on inflation for 2 μm and 3 μm MBs whilekeeping the PFB volume ratios between NDs and MBs constant.

FIG. 19 includes bright-field microscopy and graphical images showingthe effect on phospholipid acyl chain length on MB inflation.

FIG. 20 includes bright-field microscopy and graphical imagesdemonstrating the impact of ND/MB targeting in the inflation level usingNon Targeted (Cy5.5-MBs+FI-NDs) and Targeted(Cy5.5-DBCO-MBs+FI-Azide-NDs) particles.

FIG. 21 includes images showing a vessel occlusion using an in vitroflow system in PBS 1× and in blood.

FIG. 22 illustrates a demonstration of MB inflation in vivo aftersubcutaneous NDs infusion.

FIG. 23 illustrates a demonstration of MB targeting and inflation invivo after intravenous NDs infusion.

FIG. 24 illustrates shows representative ND and sub-micron MBsizedistribution and representative bright field microscopy image ofsub-micron MBs before the addition of NDs.

FIG. 25 illustrates representative pictures of ND-MB mixtures atdifferent time intervals.

FIG. 26 illustrates graphical results of video intensity versus time fora suspension of NBs was prepared in PBS.

FIG. 27 shows representative ND and NBsize distribution measured by DLSand qNano Gold Tunable Resistive Pulse Sensing counter system.

FIG. 28 illustrates a table of initial MBs, NBs and NDs size andconcentration used in testing.

FIG. 29 shows representative MBsize distribution measured by aMultisizer 4 Coulter counter system.

DETAILED DESCRIPTION OF THE INVENTION

An overview of exemplary embodiments of the present disclosure will bepresented initially, followed by further discussion of specific aspects.

Referring initially to FIG. 1, a flowchart 100 illustrates steps in amethod that can be used in ultrasound imaging techniques. In thisembodiment, step 110 comprises providing bubbles to the region ofinterest. In certain embodiments, the bubbles may be nanobubbles ormicrobubbles with an average bubble diameter between around 200-600 nm(nanobubbles) and 1-10 μm (microbubbles).

To circumvent the challenges of ADV for effective gas embolotherapy, theinventors took inspiration from a biological process and the second lawof thermodynamics: the process by which PFC is eliminated from the bodyby exhalation (25). When PFC liquid emulsions or PFC gas-filled MBstraverse the alveolar capillaries, PFC transfers to the air-filledalveolar space because of the large partial pressure gradient (1). Inaddition, since liquid PFC in NDs is at a much higher partial pressureand interfacial pressure (assuming equal interfacial tension) than itsgaseous counterpart in the MBs, the inventors hypothesized that whenmicron-scale or sub-micron-scale MBs are in close proximity to nanoscaleliquid PFC NDs, PFC vapor transfers from the NDs to MBs and expands them(Laplace Law). Although PFC solubility in water is low, it is notnegligible. The inventors speculate that PFC molecules would diffuse outof NDs, move freely between water molecules surrounding the NDs, andreach the adjacent MB core, as vaporized PFC has higher entropy than itsliquid state. A schematic representation of the nanodroplet-microbubblegas exchange shown is in FIG. 10 (where σ=interfacial tension). Thisexpansion phenomenon, also aided by the transfer of air from plasma dueto the osmotic gradient (13), results in large MBs that can embolizevessels without US activation. There are a few examples of not fullyunderstood phenomena in the literature that the inventors believe theinventors can partly explain with aspects of the present disclosure.Fowlkes et al. demonstrated optically vessel occlusion through ADV usingintravital microscopy on the rat cremaster muscle (22). In this study,the size of lodged MBs was bigger than the predicted 5× increase indiameter, as 2 μm diameter droplets vaporized into 76 μm mean length×36μm mean diameter or 25 μm mean length×11 μm mean diameter, respectivelywhen the vaporization was triggered in the capillaries (4-7 μm) or in alarger arteriole (˜125 μm). While the authors hypothesized that MBscoalescence will happen more in capillaries where the flow velocity issmaller than in the feeder vessel, the inventors also believe that PFCtransfer from unvaporized NDs contribute to MB inflation. In the clinic,the first heterogenous delayed enhancement of the liver due to theformation of larger MB after injection of contrast agents (Levovist andEchogen) has been reported by Wolf et al. with no precise causes normechanism (26). Interestingly, the phenomenon was described by otherswith different gas and shell types. Cardinale et al. hypothesized thatMBslowing down in the hepatic sinusoids inflated with gas from theintestinal microcirculation by osmotic effect (27), as the effect wasnot observed when patients received a second dose 24 h later (26, 27) or9 days later (27). The inventors believe that this new US-based approachwill lead to the development of new tools to better treat and detectdisease. Of the many possible applications of this novel platform, whichis beyond the scope of this report, the inventors believe the inventorscan induce microvascular embolization as was originally intended forADV, but at a much smaller dose and without the need for USvaporization. Starving cancer cells to death by restricting their bloodsupply was proposed nearly 50 years ago. Recently, systemic treatmentwith anti-angiogenesis therapy that is directed against immature tumormicrovessels (28) has offered short term benefits with limited impact onpatient survival, mostly because it has inhibited tumor growth ratherthan killing established ones. In addition, some major concerns remainsuch as tumor resistance, and side effects due to the inhibition ofnormal angiogenesis. Alternatively, mechanical occlusion orembolization, although less discriminant, has been used effectively totreat hepatocellular and renal cancers. This procedure is achieved byintroducing plain or drug eluting embolic material through a catheterthat is advanced as close as possible to the tumor feeding. Whileeffective, this procedure is not tumor-specific, is invasive and carriesthe typical risks linked to angiography. In addition, because tumors canparasitize other arteries, embolization may be incomplete. As of today,there is no effective strategy to achieve total occlusion of the tumormicrovasculature, particularly in a non-invasive manner. Expanding MBsusing droplet vaporization to microembolize tumor vasculature withoutthe need for acoustic activation has the potential to be translated inthe clinic.

In the embodiment shown in FIG. 1, method 100 comprises an aspect 110 ofproviding bubbles to the region of interest. In certain embodiments, thebubbles may have an average bubble diameter between around 200-600 nm(nanobubbles) and 1-10 μm (microbubbles). Method 100 also comprises anaspect 120 of providing nanodroplets to the region of interest. Incertain embodiments, the bubbles in aspect 110 and the nanodroplets inaspect 120 can be introduced to the region of interest via sequentialinjections (e.g. an initial injection of bubbles, followed by asubsequent injection of nanodroplets). In specific embodiments, thenanodroplets may comprise a perfluorocarbon (PFC). Method 100 furthercomprises an aspect 130 of increasing the average bubble diameter of thebubbles while the bubbles are in the region of interest. As explained infurther detail below, the average diameter of the bubbles can beincreased (e.g. the bubbles can be further “inflated”) via the transferof perfluorocarbons and phospholipids from the nanodroplets to thebubbles. In certain embodiments, the volume of the bubbles can beincreased by up to 6 orders of magnitude. Method 100 also comprises anaspect 140 of optionally exposing the region of interest to anultrasound stimulus (e.g. in order provide imaging for the region ofinterest). The increased average bubble diameter discussed in aspect 130can yield improved images by providing a stronger response signal to theultrasound stimulus.

Referring now to FIG. 2, an overview of another method 200 for providinga therapeutic effect to a region of interest. Method 200 comprisesaspects 210, 220, and 230 that are generally equivalent to aspects 110,120 and 130 previously discussed in method 100 above. Accordingly, forsake of brevity, the specifics of these aspects will not be repeatedhere. Method 200, however, further comprises an aspect 240 of providinga therapeutic effect to the region of interest. In certain embodiments,the therapeutic effect can be provided by utilizing the increaseddiameter of bubbles with a specific tumor target to achieve occlusion ofthe tumor microvasculature, effectively reducing the blood flow to thetumor.

Referring now to FIG. 3, an overview of a method 300 is provided thatcombines aspects of imaging method 100 and treatment (e.g. providingtherapeutic effect) method 200. Specifically, aspects 310, 320 and 330are equivalent to aspects 110, 120 and 130 previously discussed inmethod 100 above, while aspect 340 comprises providing a therapeuticeffect to the region of interest and aspect 350 comprises optionallyexposing the region of interest to an ultrasound stimulus (e.g. for usein ultrasound imaging). Referring now to FIG. 4, an overview of a method400 is provided for increasing an average bubble diameter, withoutspecification to imaging or therapeutic aspects. This embodimentcomprises aspects 410, 420 and 430 that are equivalent to aspects 110,120 and 130 previously discussed in method 100 above.

Aspects of the present disclosure also relate to methods where theincreased buoyancy of the bubbles can be used to isolate bubbles andtargeted cell structures. Referring now to FIG. 5, a method 500 includesan aspect 510 that comprises obtaining a blood sample that containsmicrobubble-cell complexes. Method 500 also comprises an aspect 520 ofproviding ex vivo nanodroplets to the blood sample. In addition, method500 includes an aspect 530 of using the ex vivo nanodroplets to increasethe average bubble diameter and buoyancy of the microbubble-cellcomplexes. Method 500 further comprises aspect 540 of retrieving cellsfrom the blood sample.

Referring back now to particular aspects of the present disclosurediscussed in FIGS. 1-4, the inventors have found that NDs act as a PFCand phospholipid reservoirs that transfer to the adjacent NBs or MBs,which triggers their growth. Interestingly, while both NBs and NDsindividually exhibit very limited ultrasound signal on B-mode imagingwhen exposed to low acoustic power, their combination results in a rapidand dramatic increase in signal visible on standard ultrasound imaging.The inventors hypothesize that the very poor water solubility and highvapor pressure of PFC allow condensed PFC from the NDs to dissipate anddiffuse away into the aqueous phase to reach the NB core and initiateexpansion. This innovation is different from the use of NDs as phasechange contrast agent (PCCA) because no sound waves are necessary toinflate the NBs or MBs.

The inventors have demonstrated rapid NB expansion when mixed with aconcentrated solution of fluorescein-labeled NDs (˜10:1 ND/NB ratio) bymicroscopy (shown in FIGS. 6 and 7). The inventors have showed evidencethat the phospholipids from the ND shell were incorporated with thenewly formed microbubbles (MBs) by fluorescence microscopy.

FIG. 6 shows representative bright field images of NDs and NBs prior andafter mixing at 20× magnification. Note that even if NDs and NBs havesimilar diameters, NDs cannot be distinguished as the refractive indexof liquid perfluorobutane is close to the refractive index of water, andimportantly, NB and ND s are not visible because of the microscoperesolution. Upon mixing on the microscopy slide, 5-20 μm MBs wereobserved instantaneously (ND+NB). Scale bar is 20 μm.

FIG. 7 shows representative bright field and fluorescence images of NDsbefore and after mixing with NBs at 20× magnification. Note that postmixing (ND+NB), newly generated MBs show higher fluorescence intensityin their shell, compared to the background fluorescence from the NDs,which indicates that fluorescein labeled phospholipids(DSPC-PEG2Kfluorescein) have been incorporated in the MBs shell. Scalebar is 20 μm.

FIG. 8 shows a schematic representation of the one-compartmentexperimental setup (left). NB expansion was observed after less than 10seconds after addition of the NDs in the NBsolution. Note that the NBsalone (0 s) only exhibit a low contrast enhancement compared to MBs (10,30 and 40 s). Mixing NBs and NDs resulted in a marked enhancement onB-mode US imaging.

FIG. 9 shows a schematic representation of the two-compartmentexperimental setup (left). Briefly, two-bulb compartments were done bycutting plastic bulbs and joining them together with a dialysis membranein between and with parafilm to seal the system. Thirty seconds afteraddition of NDs in the upper compartment (right on ultrasound), NBs inthe lower compartment (left on ultrasound) expanded to MBs producing ahigh contrast-to-noise ratio on B-mode US imaging. As expected, noacoustic droplet vaporization was observed in the upper ND compartmentas they were exposed to the lowest ultrasound transmit power (MI=0.05,PNP=0.13 MPa.

Referring now to aspects of the present disclosure that relate toimprovements on the current cell isolation buoyancy method usingtargeted MBs, the inventors have demonstrated in vitro that the additionof liquid PFC NDs (300 nm) to a MB (0.5-10 μm) suspension dramaticallyexpands the gas bodies by up to 6 orders of magnitude, and does sowithout direct contact. NDs act as a PFC and phospholipid reservoirsthat transfer to the adjacent NBs or MBs to trigger their growth.According to one aspect of the present disclosure, the number ofattached MBs per cell required to induce buoyancy and cell recoverycould decrease significantly, as only one attached MB could potentiallygrow to become 100 μm. This aspect can improve upon the buoyancytechnique used to isolate cells. It can also be applied in vivo, byadministering the NBs or MBs intravenously targeted to a circulatingcell surface receptor of interest, and then adding the liquid PFC to anextracted blood sample to detect the targeted cells. Aspects of thepresent disclosure can improve upon existing technology in at least twoways: (1) Increasing buoyancy using PFC transfer from superheated PFCNDs will decrease the number of MB per cell needed to cause cells tofloat; and (2) targeted NBs that are more effective at locating theirtarget both in vitro and particularly in vivo, and allow theaccumulation of more NBs at the cell surface, can also induce attachedcells to float when they are inflated by the liquid PFC.

The current standard for cell isolation is to target magnetic beads tothe cell surface receptor of interest that can only be done in vitro andrequires special equipment for isolation and then cell handling toremove the magnetic beads. The use of buoyancy to isolate cells improvesupon the magnetic bead strategy by not requiring MB removal afterisolation, simplifying the isolation technique, and adding the potentialof administering the MBs intravenously to search for circulating cellsof interest prior to isolation.

Aspects of the present disclosure can provide a solution to when notenough MBs attach to the cell of interest to make it buoyant, or if NBsare used to improve cell interaction but the total gas bodies remaininsufficient to cause attached cells to float. When a blood sample orany cell or particle suspension that contains the NBs or MBs attached tocells or particles that need to be isolated is spiked with liquid PFC,preferably as an emulsion of superheated PFC ND such as PFB ND that maybe targeted or non-targeted to the attached NBs or MBs, the gas bodieswill inflate because of their lower PFC pressure, increasing buoyancyand causing the attached cells/particles to float. This new strategyshould improve the detection limits of cells or particles in anysuspension.

With typical existing techniques, the buoyancy of cells or particles tobe detected or isolated depends on the number and size of MBs attachedto their surface to overcome the gravitational force exerted on theattached cell or particle. This can be a limitation that severelyimpacts detection sensitivity when using buoyancy for isolation. Aspectsof the present disclosure addresses this problem and improves thesensitivity of isolation by simply adding liquid PFC, preferably asemulsion of superheated PFC ND such as PFB ND, to inflate the attachedMBs or NBs, increasing the force to overcome the gravitational forceincreasing buoyancy and improving cell isolation.

Unlike magnetic bead isolation, aspects of the present disclosure can beused in vivo to search for the rare circulating cells, and do notrequire cell manipulation to remove the beads after they are isolated.Unlike current buoyancy techniques, aspects of the present disclosurewill allow the use of NBs that are more efficient at targeting, and canovercome the gravitational force for large cell or particle masses, byinflating the gas bodies attached to the cell or particle surface. Fortherapy, there are several schemes that this technology offers thatcould be exploited. These are merely some potential scenarios out ofseveral. One potential method is the ease by which a large payload canbe placed either within or attached on the shell of the droplets ascompared to microbubbles. These droplets either passively or activelytargeted or by merely passing through the site of interest carried byblood can be induced into phase-transition by the variety of techniquesmentioned above.

Further description and explanation of the operating principles can alsobe found in the discussion of the example and results that follow.

V. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the disclosure. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the disclosure, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe disclosure.

The important number of diseases diagnosed at a late stage because ofabsence of symptoms is objective evidence that there is a need to extendthe toolbox of imaging agents to improve early detection of disease.This work demonstrates for the first time that the addition of liquidPFC NDs to a suspension containing MBs or nanobubble (NBs) leads to thedramatic expansion of the gas bodies by at least 2 and up to 6 orders ofmagnitudes and does so without requiring direct contact between NDs andMBs or NBs. Additional advantages of our method compared to ADV is thatsmaller droplets not only exhibit greater stability as they are subjectto higher Laplace Pressure but will also ease inflation as thedifference in interfacial pressure between bubbles and droplets will behigher. Conversely, it has been reported that ADV requires more acousticpower to trigger the vaporization of smaller the NDs. Targeted NBs havebeen proposed to improve tumor detection, since they share some of theadvantages of NDs. However, their limited reflectivity at clinicalfrequencies remains an issue. By chasing targeted NBs or small MBs withNDs, bubbles will expand, thus enhancing their ultrasound signal andimproving the detection of the targeted diseased tissue. The inventorsvalidated this first milestone in vivo, using 1 μm rhodamine-labeled MBsthat target MC38 cancer cells in a tumor-bearing mouse.

Nanobubbles Inflate when in Contact with Nanodroplets

As a proof of concept, the inventors first used perfluorobutane (PFB)nanobubbles (NBs) instead of regular microbubbles (>1 μm) to explore theeffect of the addition of PFB NDs on the signal intensity by ultrasoundimaging. NBs constitute a good model because of their limited US signalon B-mode imaging when exposed to low acoustic power. Both NBs and NDswere prepared with a phospholipid mixture composed of1,2-Distearoylphosphatidylethanolamine (DSPC) andDistearoylphosphatidylethanolamine conjugated with polyethylene glycol2000 (DSPE-PEG 2K) in a 90:10 molar ratio. Their hydrodynamic sizes werecharacterized by dynamic light scattering (DLS, Figure S1A) and were onaverage 488.1±4.6 nm and 285.2±0.9 nm with polydispersity indexes (PdI)of 0.20±0.004 and 0.17±0.003 for NBs and NDs respectively. Tunableresistive pulse sensing (TRPS) was used to measure their size andconcentration (FIGS. 29 and 30). FIG. 24 shows (A) Representative ND andsub-micron MB size distribution measured by DLS. (B) Representativebright field microscopy image of sub-micron MBs before the addition ofNDs. Scale bar=100 μm. FIG. 27 shows representative DLS (A) and TRPS (B)traces of nanodroplets and nanobubbles (n=3). FIG. 29 shows multisizercoulter counter traces of MBs (n=3).

A suspension of NBs was prepared in PBS 1×(3×10⁹ NBs/mL) and introducedat the bottom of a plastic bulb with the intent of observing any changein buoyancy and echogenicity. The plastic bulb was then immersed in awater bath at 37° C. and imaged using B-mode imaging. US imaging at lowmechanical index (MI=0.05) produced only a weak signal despite highconcentration of NBs (FIG. 11A). Once the temperature of theNBsuspension reached 37° C., a suspension of NDs in PBS 1×(10⁹ NDs/mL)was added in the plastic bulb. After a few seconds, the ultrasoundsignal increased along with the appearance of numerous echogenic andbuoyant MBs that started to rise to the surface. Regions of interest(ROIs) were drawn and the ultrasound video signal intensity in theplastic bulb was measured and resulted in a 4-fold enhancement afteronly 30 s post-ND addition (FIG. 24A and FIG. 26). Interestingly, theportion of the bulb located in the far field is not visible after 45 s,due to the acoustic shadowing that resulted from the high number of MBsformed by this inflation process. As expected (29), NDs alone did notproduce any US signal when observed at low MI for 5 minutes (FIG. S4)and vaporized only when subjected to a MI=0.5. NB inflation was alsoobserved optically by microscopy using the same samples used in theultrasound experiment. Briefly, a small volume (10 μL) of NBs in PBS 1×(1×10¹¹ NBs/mL) was first placed on a microscopy slide and covered witha coverslip and a small volume of NDs (10 μL) in PBS 1×(1×10¹¹ NBs/mL)was then added between the slide and the coverslip to mix NBs and NDstogether. The mixture was observed using bright filed microscopy at a20× magnification. The first large MB was formed after only 2 secondsand more inflated MBs were formed subsequently with sizes above 50 μm(FIG. 11B). FIG. 11 illustrates PFB nanobubbles inflation triggered bythe addition of PFB nanodroplets. A) B-mode ultrasound imaging of NBsbefore, immediately after, and 2, 5, 10, 15, 30 and 45 s after NDaddition in PBS 1× at 37° C. B) Bright-field microscopy time lapse of NBinflation after ND addition. Scale bar is 100 μm

Microbubbles Inflate without Contact with Nanodroplets

While the two previous experiments provided evidence of inflation whenNBs and NDs are mixed together in solution, they did not demonstrate ifdirect contact between NB and ND is necessary to trigger expansion. Inaddition, as both systems are in equilibrium with ambient pressure, thelarge expanded MBs have the ability to draw more air into them fromoutside and expand even more. The inventors expect that similar MBinflation will happen in plasma with the dissolved air.

To assess if direct contact is necessary to trigger inflation, theinventors performed two additional experiments using ultrasound imagingand microscopy. In a first experiment, two soft plastic bulbs halveswere attached to each other, their inner compartments separated with asemi-permeable dialysis membrane (3.5 kDa molecular weight cutoff, 60 μmthickness). The lower compartment was filled with a suspension of PFBNBs in PBS 1×, while the upper compartment was first filled with PBS 1×.The two-compartment sample holder was then placed in a water bath at 37°C. and was imaged by ultrasound at low MI (MI=0.05) (FIG. 12A). Asuspension of PFB NDs was then added in the upper compartment. Thesemi-permeable membrane allows small molecules such as PFB orphospholipids to freely diffuse while preventing NDs and NBs fromdiffusing from one compartment to the other. Using on B-mode imaging at8 MHz, no signal was visible in the lower compartment containing NBs.Interestingly, less than 1 min after the addition of NDs in the uppercompartment, a significant ultrasound signal was observed in the lowercompartment, characteristic of echogenic microbubbles being inflated.(FIG. 12A). Samples before and after addition of NDs were collected andobserved under the microscope to visually assess NB expansion.Bright-field microscopy showed a large amount of expanded MBs with manyaround 50 μm in size (FIG. 12B). FIG. 12 shows validation of MBexpansion in the presence of NDs without direct contact using ultrasoundimaging and optical microscopy. A) Schematic representation of theultrasound setup using two compartments separated by a semi-permeablemembrane and B-mode US imaging before (left) and after (right) theaddition of NDs in the upper compartment. B) Representative bright-fieldmicroscopy images of NBs in the lower compartment before (left) andafter (right) addition of NDs in the upper compartment.

In a second experiment, an optically transparent two-compartment systemseparated by a semi-permeable membrane (MWCO=3.5 kDa, 60 μm thickness)was built between a microscopy slide and a coverslip to observe MBinflation in real time using bright-field microscopy (FIG. 13A). AMBsuspension in PBS 1× was placed in the lower compartment and eitherPBS 1× or a suspension of NDs in PBS 1× were added in the uppercompartment. The focal plane was adjusted to observe MBs in the lowercompartment during the addition of PBS 1× or NDs in the uppercompartment. As expected, no expansion was observed when PBS 1× wasadded while the formation of 50 μm inflated MBs was observed when NDswere added after around 5 minutes (FIG. 13B). FIG. 13 illustrates anobservation of MB expansion in the presence of NDs without directcontact using microscopy. Top: Schematic representation of thetwo-compartment system containing MBs in the lower compartment andeither PBS 1× (A) or NDs (B) in the upper compartment. Bottom:Representative bright field images of MBs in the lower compartment afteraddition of PBS (A) or NDs (B) in the upper compartment. Scale bar is100 μm.

Membrane Fusion Occurs Between NDs and MBs During Inflation.

To assess if any membrane fusion occurs during the inflation process,NDs and MBs formulated using phospholipids labeled with Cy5.5(Cy5.5-MBs) or fluorescein (Fl-NDs) respectively. Flow cytometry wasthen used to evaluate the fluorescence of the MBs after addition ofFl-NDs (FIG. 5A). ROIs P3 and P4 were drawn on the scatter-plots toidentify NDs and MBs respectively. Gating the MBs (P4 gate), a bivariatehistogram was generated with four quadrants for the four possiblefluorescence combinations (F1−/Cy5.5−, Fl+/Cy5.5−, Fl−/Cy5.5+,Fl+/Cy5.5+). While MBs were initially Fl−/Cy5.5+ in the lower rightquadrant (99.8% in Q4-LR), the addition of Fl-NDs caused a shift of theMBsignal to the Fl+/Cy5.5+ upper right quadrant within a minute (97.7%in Q4-UR), which indicates that Fl-labeled phospholipids from NDs wereincorporated into the MB phospholipid shell, thus demonstrating membranefusion. Finally, a P4-gated histogram representing the particle count infunction of forward scatter (FSC) demonstrated MB expansion as 81.3% ofthe inflated MBs were bigger than the largest MBs prior to ND addition.

As MBs kept inflating overtime (FIG. 25), larger MBs started toaccumulate to the surface over time to form a foamy upper layer. Inaddition, as these expanded MBs are saturated in PFC and the sample isin equilibrium with ambient pressure, expanded MBs most likely pulledair from the surface to expand even more. Because of their extremebuoyancy, the number of inflated MBs is underestimated by flowcytometry, as a large population of inflated MBs is not injected in theflow cytometer since its inlet is located at the bottom of the sampletube. In addition, MBs>40 μm are not detected as they are above thedetection limit of the instrument. Because of these limitations, thequantitative analysis of the expansion of MB with ND was not possibleusing flow cytometry. FIG. 25 shows representative pictures of the ND-MBmixtures. The foam observed on top of the Eppendorf are characteristicsof large buoyant MBs that float to the top.

To confirm the flow cytometry data, the inventors also used fluorescencemicroscopy to visualize membrane fusion by observing the incorporationof lipids from the NDs shell in the inflated MBshell. In a firstmicroscopy experiment, the inventors labeled the phospholipid membranesof MBs and NDs with the lipophilic tracers DiD(1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine) and DiO(3,3′-dioctadecyloxacarbocyanine) respectively. Addition of DiO-labeledNDs in a suspension of DiD-labeled MBs lead to the formation of inflatedMBs exhibiting both DiO and DiD fluorescence (FIG. 5B), providingfurther evidence of a membrane fusion phenomenon.

To provide further insight in the inflation mechanism, an experimentalsetup identical to that described for FIG. 13 was employed but usingCy5.5-MBs and Fl-NDs at similar concentrations. Surprisingly, while NDsand MBs were separated by a 60 μm thickness membrane with MWCO of 3.5kDa, the inventors found that DSPE-PEG2K-fluorescein (MW=2.4 kDa) wereincorporated in the inflated shell of the MBs within minutes (FIG. 14C).These results suggest that expanded MBs formed with our process will bemost likely more stable than MBs generated from ADV. FIG. 14 shows ademonstration of membrane fusion between NDs and MBs during inflation.A) Flow cytometry data of Cy5.5-MBs alone (top) and Cy5.5-MBs mixed withFl-NDs (bottom) including a scatter-plot (left) with ROIs drawn for MBs(P4) and NDs (P3), a P4-gated bivariate histogram (center) with fourquadrants for the four possible fluorescence combinations (F1−/Cy5.5−,Fl+/Cy5.5−, Fl−/Cy5.5+, Fl+/Cy5.5+), and P4-gated particle count infunction of forward scatter (higher forward scatter=larger size). B)Representative bright field and fluorescence microscopy images showingcolocalization of DiO-NDs and DiD-MBs. Red boxes highlight coalescenceand fusion of MBs observed between acquisitions of bright field andfluorescence images. C) Validation of fluorescein labeled phospholipidtransfer through the dialysis membrane (3.5 kDa cutoff, 60 μmthickness). top) Schematic representation of the two-compartment systemwith Fl-NDs and Cy5.5-MBs. bottom) Representative bright field image ofinflated MBs in the lower compartment that now contains Fl-DSPE-PEG.Scale bar is 100 μm.

To assess whether a phospholipid membrane was needed to induce MBinflation, the inventors also investigated the ability of MBs with ashell composed of denatured albumin to expand when in contact withphospholipid based NDs. Albumin-based MBs are formulated by tipsonication of albumin in the presence of PFB. The heat generated bysonication denatures and crosslinks albumin via disulfide bonds to forma stiff shell around the PFC core. The inventors hypothesized that apolymer-based or crosslinked proteins shell may physically limitbubbles' growth, as lipid surfactants provided by NDs should notcontribute and promote further inflation. To observe MB inflation inreal time, the inventors place a suspension of albumin-MBs in ahemocytometer plate and added a suspension of PFB-NDs. Interestingly,albumin-MBs inflated at a fast rate, exhibiting MBs>10 μm immediatelyafter addition of PFB phospholipid NDs (FIG. 15A, B). However, themaximal size reached by inflated albumin-MBs was limited to an averageof 23±x μm 30 minutes after ND addition (FIG. 15C), which is smallerthan sizes observed in our previous experiments with phospholipid-basedMBs. This experiment further validated our hypothesis that PFC transferoccurs without membrane fusion and despite the different shellcompositions between NDs and MBs. FIG. 15 shows inflation occurs whenAlbumin MBs are mixed with phospholipid PFB NDs. (A) Bright-fieldmicroscopy images of albumin MBs over time. B) Number and C) averagesize of inflated albumin-MBs over time. Scale bar is 100 μm.

Lower Molecular Weight PFCs in ND Core Induce Higher Count and Faster MBInflation.

Nanodroplets described herein were formulated by microfluidization usingeither PFB, perfluoropentane (PFP) or perfluorohexane (PFH) as liquidcore with phospholipid shells containing DSPC and DSPE-PEG2K with a90:10 molar ratio. PFB MBs were prepared with the same phospholipidcompositions so they all share the same interfacial tension. All NDsamples were characterized by DLS and their hydrodynamic diameters wereon average 221±4 nm (PFB), 295±1 nm (PFP), and 268±5 nm (PFH) (FIG. 28).ND size and concentration were measured using TRPS (FIG. 27 and FIG.28). Both DLS and TRPS data confirmed that the ND size was not affectedby the nature of the encapsulated PFC, which was expected asmicrofluidization using high shear fluid processors is an efficienttechnique to reduce and achieve uniform emulsion size. PFB NDs yieldedthe largest amount of inflated MBs and exhibited the highest inflationrate. PFP ND yielded some inflation after 10 min and inflated MBs to alesser extent with no MB>40 μm. Finally, PFH NDs did not yield any MBinflation. This result validated our hypothesis that lower molecularweight PFC are transferred more efficiently into MBs, most likely as aresult of their lower intermolecular cohesiveness, higher PFC vaporpressure (PFB: 330.0 kPa, PFP: 84.0 kPa, PFH: 31.0 kPa) and volatility(30). FIG. 16 shows the effect of ND PFC core on PFB MBs inflation. A)Representative bright-field microscopy images showing MB inflation afterthe addition of perfluorobutane (PFB), perfluoropentane (PFP), orperfluorohexane (PFH) NDs (scale bar=100 μm). B) Number and C) averagesize of inflated MBs over time.

Large MBs Inflate Faster than Small MBs or NBs.

The inventors' hypothesis is that the larger MBs will be inflated at afaster rate compared to smaller MBs or NB due to the greater differencein Laplace pressure in regard to NDs (assuming equal interfacialtension). For these reasons, the inventors tested the inflation rate ofbubbles with different mean sizes (3 μm, 2 μm, and 0.3 μm) when combinedwith NDs at a ND/MB ratio of 100:1. Both droplets and bubbles wereformulated with the same phospholipid shell composition (DSPC:DSPE-PEG2Kat a 90:10 molar ratio). When mixed with NDs, the larger 3 μm MBsstarted to expand immediately after ND addition, while 2 μm MBs and 0.3μm NBs started to inflate 5 and 10 min after ND addition respectively(FIG. 17A). Assuming equal surface tension, 3-μm MBs are under lowerinterfacial tension than 2-μm MBs and 0.3-μm NBs. According to Laplacelaw, the inventors hypothesize that as liquid PFC in NDs exhibit a muchhigher interfacial pressure difference with big MBs, PFC transfer willhappen faster with 3-μm compared to 3-μm and 0.3-μm MBs. In addition,the inventors assume that, at similar NDs to MBs ratio, the opportunityfor the NDs to interact with NBs and transfer its PFC and phospholipidsis lower due to a decreased probability to be close to each other.Interestingly, both NBs and MBs continued to inflate over 30 minreaching nearly similar size. This is in agreement with the inventors'theory that the large vapor pressure in the NDs is the main drivingforce, as the concentration gradient is expected to be the same whateverthe bubble size. FIG. 17 shows the effect of bubble size on theirinflation. A) Representative bright-field microscopy images showinginflation of 3 μm, 2 μm and 0.3 μm MBs after the addition of PFB NDs ata 100:1 ND/MB ratio (scale bar=100 μm). B) Number and C) average size ofinflated MBs over time.

In order to better compare the effect of MBsize on inflation, theinventors also compared the inflation of 2 μm and 3 μm MBs while keepingthe PFB volume ratios between NDs and MBs constant, as opposed tokeeping a 100:1 NB/MB concentration ratio. This experiment exhibited asimilar trend compared to the previous experiment with larger 3 μm MBsbeing inflated at a faster rate than 2 μm MBs (FIG. 18). FIG. 18 showsMB inflation of 2 μm and 3 μm MBs while keeping PFC volume ratio betweenND and MB constant. A) Representative bright-field microscopy images ofinflation over time. B) Number and C) average size of inflated MBs overtime. Scale bar is 100 μm.

MB and ND Membrane Compositions Dictates MB Inflation Kinetics.

It is known that the transition temperature of phospholipids increaseswith the acyl chain length. This causes MBs formulated usingphospholipids with longer acyl chains to have more cohesive shells. Thisincrease in shell cohesiveness should delay the transfer of PFC gasacross the MB membrane due to the increase in attractive hydrophobic andvan der Waals interactions between the adjacent phospholipids'hydrophobic tails. For these reasons, the inventors performed a seriesof experiments to explore the impact of the intermolecular forcesbetween phospholipids in MB and ND shell on MB inflation. Specifically,the inventors compared the rate and extent of inflation using MBs andNDs made from DSPC (18 carbons in each acyl chain and 0 insaturation,18:0) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, 16:0). AllMB/ND combinations were tested at a 100:1 ND/MB ratio. The inventorsselected these two phospholipids because while they are both common MBconstituents, they are in two different lipid physical state at 37° C.,either in their ordered gel phase (lipid chains extended and closelypacked) or fluid phase (lipid chains randomly organized) for 18:0 and16:0 respectively. As expected, MBs made with 16:0 phospholipidsunderwent a faster and more important inflation than their analogueswith 18:0 phospholipids, and so with NDs composed of either 18:0 or 16:0phospholipids (FIG. 19). This result is most likely due to the easiergas penetration through the monolayer composed of phospholipids withshorter acyl chains (32).

While less lipid intermolecular cohesion forces in NDs made of 16:0 vs.18:0 phospholipids are known to result in lower activation energy neededto acoustically trigger ND vaporization (33), our results showed 18:0MBs inflated to a greater extent with 18:0 NDs as opposed to 16:0 NDs.While the inventors do not have an indisputable explanation, theinventors hypothesize that the fact that 18:0 NDs were smaller thantheir 16:0 counterpart compensated their lower interfacial tensionresulting in similar or close Laplace overpressure. This result may alsobe explained by the presence or absence of surface microstructures thathave been reported in MBs composed of DSPC and DPPC respectively (34).The inventors hypothesize that similarly to MBs, 18:0 NDs have surfacemicrostructures whereas 16:0 NDs do not, which explain their morefavorable phospholipids and gas transfer. FIG. 19 shows the effect onphospholipid acyl chain length on MB inflation. A) Representativebright-field microscopy images of MB inflation using MBs/NDs with 18:0(DSPC) and 16:0 (DPPC) phospholipids lengths (scale bar=100 μm). B)Number and C) average size of inflated MBs over time.

Targeting NDs to MBs Through Bioorthogonal Click Chemistry FacilitatesMB Inflation.

To prove the effect of targeting on non-acoustic droplet vaporization,the inventors used bio-orthogonal click chemistry, a copper-free clickchemistry reaction between an azide and a strained alkyne (e.g.,cyclooctyne) that has been used in vivo for imaging (35, 36) or therapy(37) with no toxicity. The inventors expect that targeting NDs to MBswill bring them into close proximity to ease PFC transfer, increaselipid fusion, and maximize inflation while minimizing dose. Theinventors used dibenzocyclooctyne (DBCO) as our strained alkyne, becauseit is commercially available already attached to a PEGylatedphospholipid and has been shown to trigger artificial membrane fusionwith azide-labeled phospholipids.(38) To demonstrate the advantage oftargeting NDs to MBs, the inventors tested a series of ND:MBconcentration ratios (1:1 to 100:1) while keeping the MB count andvolume constant. MB inflation was assessed by flow cytometry asdescribed above, using Cy5.5-MBs and FI-NDs (Non targeted) as well asCy5.5-DBCO-MBs and FI-Azide-NDs. Below 100:1 ND:MB ratio, the inventorsdid not observe any MB inflation with or without targeting at theconcentrations tested. However, at a 100:1 ND:MB ratio, while only12.1±2.1% MBs inflated in the non-targeted samples, 40.2±16.4% DBCO-MBsinflated (FIG. 20A). The inventors hypothesized that non-targeted NDmost likely did not interact with non-targeted MBs as much as theytargeted analogues which resulted in a lower inflated MB count. Even ifthe previous experiment described above were in agreement with an effectof targeting on MB inflation, the inventors believe that this effect isunderestimated due to the flow cytometry limitation mentioned earlier.To provide further evidence of the enhanced MB inflation when MB and NDare targeted to each other's, the inventors evaluated by microscopy MBinflation in the presence of NDs using disposable hemocytometers (FIG.20B). Contrary to the flow cytometer, this microscopy study does notsuffer from MB buoyancy or increased size. FIG. 20 shows a demonstrationof the impact of ND/MB targeting in the inflation level usingNon-Targeted (Cy5.5-MBs+FI-NDs) and Targeted(Cy5.5-DBCO-MBs+FI-Azide-NDs) particles. A) Left: Flow cytometryscatter-plots with ROIs drawn for MBs (P4) and NDs (P3), and P4-gatedbivariate histograms with four quadrants for the four possiblefluorescence combinations (Fl−/Cy5.5−, Fl+/Cy5.5−, Fl−/Cy5.5+,Fl+/Cy5.5+) of Non Targeted (left) and Targeted (right) at 100:1(bottom) and 1:1 (top) concentration ratios. A) Right: Percentagecolocalization between Non-Targeted and Targeted at P4-gated bivariatehistogram in the upper right quadrant (F1+/Cy5.5+) as a function ofincreasing ND:MB concentration ratios. B) Left: Representativebright-field microscopy images of MB inflation using Non targeted andTargeted MBs/NDs (scale bar=100 μm). B) Right: Number of inflated MBsover time.

Immobilized Microbubbles Inflate when in Contact to Nanodroplets in FlowConditions in PBS 1× and in Blood.

To evaluate the potential of the approach for in vivo applications, theinventors performed in vitro experiments in a flow system using PBS1×and whole blood. Since flow conditions will render interactions betweenND and MB more challenging, the inventors assessed the effect of flow onMBs/NBs inflation in order to validate their formulations and inflationconditions prior to in vivo experiments. To mimic blood vessels and cellreceptors, the inventors used a medical polyimide tubing (200 μm I.D.)coated with fluorescein. This coating was done by filling the tube withethylenediamine and then conjugating the free amines with NHS-FITC usingEDC as a coupling agent as previously reported (39). For the experimentin PBS1×, MBs were composed of DSPC, DSPE-PEG and DSPE-PEG-maleimide ata 90:8:2 molar ratio. NDs were composed of DSPC, DSPE-PEG, DSPE-PEG-Maland DSPE-PEG-FITC with a 90:6:2:2 molar ratio. For the experiment inblood, lipid compositions were identical for both MBs and NDs but MBs.In order to mimic receptor targeting, MBs were conjugated with ananti-FITC antibody. Briefly, anti-FITC antibody was first thiolatedusing 2-iminothiolane, purified through a desalting column, and reactedwith MBs via a thiol-ene coupling reaction. Targeted MBs (10⁷/mL), inPBS 1× or in blood, were infused through the tube at a flow ratemimicking capillarity velocity (0.01 cm/s, 0.19 μL/min for 5 min) andthen washed with PBS 1× or blood to remove unbound MBs (manualinfusion). Bound MBs were easily visible after the wash in PBS but notin blood (FIG. 21A-top in PBS, data not shown in blood) by microscopy.Fluorescein-labeled NDs (Fl-NDs) were infused at the same flow rate. MBinflation and tube occlusion occurred within seconds after NDs infusion(10¹⁰/mL) in PBS (FIG. 21 A-bottom), with MB fusion observed in realtime upon contact. In blood, the inventors observed a few MB inflatedbelow a minute, however no coalescence was observed between inflated MBsuntil 15 min of ND infusion. Partial blood flow occlusion was observedafter 15 min (red blood cell accumulation observed upstream from theocclusion site via darkening of the tube) and blood flow cessation wasobserved after 30 min (FIG. 21B). Note that after 30 min, a blood clotformed in the tube because of lack of flow. Blood flow was recovered byincreasing the syringe pump volume dramatically (10 μL/min, >5.2 mm/s).This delayed onset of both inflation and fusion observed in blood wasexpected because of the higher blood viscosity that likely slightlydecrease PFC gas kinetics transfer and stabilize inflated MBs towardscoalescence. FIG. 21 shows a vessel occlusion using an in vitro flowsystem in PBS 1× and in blood. A) Representative bright field (left) andfluorescence (right) microscopy images of MBs before (top) and after(bottom) NDs infusion. B) Representative bright field of MBs during NDsinfusion in blood.

In Vivo Validations of Microbubble Expansion.

The inventors first injected 10⁶ MBs diluted in 10 uL PBS into thesubcutaneous space of a mouse skin flap to assess if MB inflation wouldoccur in vivo. 5 min post MB injection (FIG. 22), 5×10⁹ NDs diluted in10 uL PBS were injected along the same tract into the subcutaneousspace. While observing the injection area, the 1 μm MBs visualized inbright-field started to expand after 30 s post ND injection. Theinventors were also able to observe both an increase in the number ofexpanded MBs, as well as continued expansion of already expanded MBs upto ˜50 μm. FIG. 22 shows a demonstration of MB inflation in vivo aftersubcutaneous NDs infusion. Scale bar is 100 μm.

The inventors then injected 2×10⁸ targeted MBs diluted in 100 uL PBSinto the right retro-orbital sinus of a MC38-bearing nude mice to assesswhether MB inflation would occur in vivo and in blood (FIG. 13).Targeted MBS were composed of DSPC, DSPE-PEG, DSPE-PEG-maleimide and1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamineB sulfonyl) (DPPE-Liss Rhod) at an 89.5:5:5:0.5 molar ratio. In order tomimic receptor targeting, MBs were conjugated with ananti-phosphatidylserine (PS) antibodies. Antibodies targeting PS are oneof the most selective strategies to target tumor blood vessels and havebeen validated in numerous animal models of cancer. After 15 min, theinventors proceeded to create a dorsal skin flap, as done previouslywith the tumor in the center of the viewing window (see FIG. 23A).

Due to their small size (˜1 μm) and most likely deep location within theattenuative tissue, MBs were not easily localized using bright field.However, the inventors were initially able to appreciate only a few somesmall, immobilized spots of rhodamine signal in the vessels in orimmediately adjacent to the tumor (FIG. 13B-C). The high background inthe Rhodamine channel (FIG. 13C) is the result of the high exposure timeneeded (300 ms) to capture a signal. At 30 minutes after the initial MBinjection (in order to allow MB clearance from the circulation by thelungs), the inventors then introduced 4×10¹¹ NDs composed of DSPC,DSPE-PEG, DSPE-PEG-maleimide in a 90:8:2 molar ratio into the leftretro-orbital sinus. NDs were conjugated with an anti-phosphatidylserine(PS) antibodies. While only a sparse number of MBs were observed pre-NDinfusion in the rhodamine channel (FIG. 23C), more MB signal wasobserved (FIG. 23D) and immediately adjacent to the tumor, around 10 minpost ND injection. The lower background is possibly due to reduction ofthe exposure time (250 ms). FIG. 23 shows a demonstration of MBtargeting and inflation in vivo after intravenous NDs infusion. A)Dorsal skin flap as prepared for microscopy with sub-centimetersubcutaneous MC38 tumor. The nose cone and isoflurane anesthesia weretemporarily removed for the purpose of taking this picture. B-C)Representative bright Field (B) and fluorescence microscopy images (C)of targeted MBs prior NDs injection. D) Representative fluorescencemicroscopy images of inflated MBs after 12 min post NDs injection. Scalebar is 100 μm.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

V. REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   1. Wu Y, Unger E C, Mccreery T P, Sweitzer R H, Shen D, Wu G,    Vielhauer M D. Binding and lysing of blood clots using MRX-408.    Invest Radio/. 1998 December; 33(12):880-5.-   2. Lindner J R, Song J, Christiansen J, Klibanov A L, Xu F, Ley K.    Ultrasound assessment of inflammation and renal tissue injury with    microbubbles targeted to P-selectin. Circulation. 2001 Oct. 23;    104(17):2107-12.-   3. Weller GER, Lu E, Csikari M M, Klibanov A L, Fischer D, Wagner W    R, Villanueva F S. Ultrasound Imaging of Acute Cardiac Transplant    Rejection With Microbubbles Targeted to Intercellular Adhesion    Molecule-1. Circulation 2003; 108:218-224.-   4. Ellegala D B, Leong-Poi H, Carpenter J E, Klibanov A L, Kaul S,    Shaffrey M E, Sklenar J, Lindner J R. Imaging Tumor Angiogenesis    With Contrast Ultrasound and Microbubbles Targeted to Alpha-v    Beta-3. Circulation 2003; 108:336-341.-   5. Hall C S, Marsh J N, Scott M J, Gaffney P J, Wickline S A, Lanza    G M. Time evolution of enhanced ultrasonic reflection using a    fibrin-targeted nanoparticulate contrast agent. J Acoust Soc Am.    2000; 108(6):3049-57.-   6. Lanza G M, Abendschein D R, Hall C S, Scott M J, Scherrer D E,    Houseman A, Miller J G, Wickline S A. In vivo molecular imaging of    stretch-induced tissue factor in carotid arteries with    ligand-targeted nanoparticles. J Am Soc Echocardiogr. 2000 June;    13(6):608-14.-   7. Mattrey R F, Steinbach G C: Ultrasound Contrast Agents: State of    the Art. Invest Radio/. 1991; 26:S5-S11.-   8. Ophir J, Parker K J. Contrast agents in diagnostic ultrasound    [published erratum appears in Ultrasound Med Biol 1990; 16:209]    Ultrasound Med Bio/1989; 15(4):319-33-   9. Tiemann K, Becher H, Bimmel D, Schlief R, Nanda N C. Stimulated    Acoustic Emission Nonbackscatter Contrast Effect of Microbubbles    Seen with Harmonic Power Doppler Imaging. Echocardiography. 1997    January; 14(1):65-70-   10. Klibanov A L, Rasche P T, Hughes M S, Wojdyla J K, Galen K P,    Wible J H, Brandenburger G H. Detection of Individual Microbubbles    of Ultrasound Contrast Agents Imaging of Free-Floating and Targeted    Bubbles. Invest Radiol 2004; 39: 187-195-   11. Hauff P, Reinhardt M, Briel A, Debus N, Schirner M. Molecular    Targeting of Lymph Nodes with L-Selectin Ligand-specific US Contrast    Agent: A Feasibility Study in Mice and Dogs. Radiology. 2004 in    press [Epub ahead of print]-   12. Jolesz F A, Hynynen K. Magnetic resonance image-guided focused    ultrasound surgery. Cancer J. 2002; 8 (Suppl 1):S100-12.-   13. Tempany C M, Stewart E A, McDannold N, Quade B J, Jolesz F A,    Hynynen K. M R imaging-guided focused ultrasound surgery of uterine    leiomyomas: a feasibility study. Radiology 2003; 226:897-905.-   14. Oleson J R, Cetas T C, Corry P M. Hyperthermia by Magnetic    Induction: Experimental and Theoretical Results for Coaxial Coil    Pairs. Radiation Research 1983; 95:175-186-   15. Reilly J P. Principles of Nerve and Heart Excitation by    Time-Varying Magnetic Fields. Annual New York Academy of Science    1992; 649:96-117.-   16. Jordan A, Scholz R, Maier-Hau K, Johannsen M, Wust P, Nadobny J,    Schirra H, Schmidt H, Deger S, Loaning S, Lanksch W, Felix R.    Presentation of a new magnetic field therapy system for the    treatment of human solid tumours with magnetic fluid    hyperthermia. J. Magn. Magn. Mater. 2001; 225:118-126.-   17. Suzuki M, Shinkai M, Honda H, Kobayashi T. Anticancer effect and    immune induction by hyperthermia of malignant melanoma using    magnetite cationic liposomes. Melanoma Research 2003; 13:129-135.-   18. Matsuoka F, Shinkai M, Honda H, Kubo T, Sugita T, Kobayashi T.    Hyperthermia using magnetite cationic liposomes for hamster    osteosarcoma. BioMagn Res Technol 2004; 2:3-8.-   19. Shinkai M, Ueda K, Ohtsu S, Honda H, Kohri K, Kobayashi T.    Effect of Functional Magnetic Particles on Radiofrequency Capacitive    Heating: An in vivo Study. Jpn. J. Cancer Res 2002; 93:103-108.-   20. Shinkai M, Le B, Honda H, Yoshikawa K, Shimizu K, Saga S,    Wakabayashi T, Yoshida J, Kobayashi T. Targeting Hyperthermia for    Renal Cell Carcinoma Using Human MN Antigen specific    Magnetoliposomes. Jpn. J. Cancer Res 2001; 92: 1138-1146.-   21. Moroz P, Jones S K. Gray B N. Magnetically mediated    hyperthermia: current status and future directions. Int. J.    Hyperthermia 2002; 18: 267-284.-   22. Jordan A, Scholz R, Wust P, Fahling H, Krause J, Wlodarczyk W,    Sander B, Vogl T, Felix R. Effects of magnetic fluid hyperthermia    (MFH) on C3H mammary carcinoma in vivo. Int. J. Hyperthermia 1997;    13:587-605-   23. Rosensweig R E. Heating magnetic fluid with alternating magnetic    field. J. Magn. Magn. Mater. 2002; 252:370-37 4-   24. Pankhurst Q A, Connolly J, Jones S K, Dobson J. Applications of    Magnetic Nanoparticles in Biomedicine. Journal of Physics D: Applied    Physics 2003; 36:R167-181-   25. Giesecke T, Hynynen K. Ultrasound-mediated cavitation thresholds    of liquid perfluorocarbon droplets in vitro. Ultrasound Med Biol.    2003; 29:1359-1365-   26. Kripfgans O D, Fowlkes J B, Miller D L, Eldevik O P, Carson P L    Acoustic droplet vaporization for therapeutic and diagnostic    applications. Ultrasound Med Biol. 2000 Sep. 26(7): 1177-89.-   27. Dayton P A, Allen J S, Ferrara K W. The magnitude of radiation    force on ultrasound contrast agents. J. Acoust. Soc. Am 2002; 112:    2183-2192-   28. Chomas J E, Dayton P A, May D J, Allen J S, Klibanov A L,    Ferrara K W. Optical observation of contrast agent destruction.    Appl. Phys. Lett. 2000; 77: 1056-1058.-   29. Fink M, Montaldo G, Tanter M. Time-reversal acoustics in    biomedical engineering. Annu Rev Biomed Eng. 2003; 5:465-97.-   30. Mattrey R F, Long D M, Peck W W, Slutsky R A, Higgins C B:    Perfluorooctyl bromide as a Blood Pool Contrast Agent for Liver,    Spleen, and Vascular Imaging in Computed Tomography. J Comput Assist    Tomogr. 1984; 8(4): 739-7 44.

ADDITIONAL REFERENCES

-   1. Mattrey R F. Perfluorooctylbromide: a new contrast agent for C T,    sonography, and M R imaging. AJR Am J Roentgenol. 1989;    152(2):247-52. doi: 10.2214/ajr.152.2.247. PubMed PMID: 2643258.-   2. Klibanov A L, Rasche P T, Hughes M S, Wojdyla J K, Galen K P,    Wible J H, Jr., Brandenburger G H. Detection of individual    microbubbles of an ultrasound contrast agent: fundamental and pulse    inversion imaging. Acad Radiol. 2002; 9 Suppl 2:S279-81. PubMed    PMID: 12188248.-   3. Klibanov A L, Rasche P T, Hughes M S, Wojdyla J K, Galen K P,    Wible J H, Brandenburger G H. Detection of individual microbubbles    of ultrasound contrast agents—Imaging of free-floating and targeted    bubbles. Invest Radiol. 2004; 39(3):187-95. doi:    10.1097/01.rli.0000115926.96796.75. PubMed PMID:    WOS:000189320200008.-   4. Cui W, Tavri S, Benchimol M J, Itani M, Olson E S, Zhang H, Decyk    M, Ramirez R G, Barback C V, Kono Y, Mattrey R F. Neural progenitor    cells labeling with microbubble contrast agent for ultrasound    imaging in vivo. Biomaterials. 2013; 34(21):4926-35. doi:    10.1016/j.biomaterials.2013.03.020. PubMed PMID: 23578557; PMCID:    PMC3742341.-   5. Lindner J R, Song J, Christiansen J, Klibanov A L, Xu F, Ley K.    Ultrasound assessment of inflammation and renal tissue injury with    microbubbles targeted to P-selectin. Circulation. 2001;    104(17):2107-12. PubMed PMID: 11673354.-   6. Leong-Poi H, Christiansen J, Klibanov A L, Kaul S, Lindner J R.    Noninvasive assessment of angiogenesis by ultrasound and    microbubbles targeted to alpha(v)-integrins. Circulation. 2003;    107(3):455-60. doi: 10.1161/01.Cir.0000044916.05919.8b. PubMed PMID:    WOS:000180786100034.-   7. Simberg D, Mattrey R. Targeting of perfluorocarbon microbubbles    to selective populations of circulating blood cells. J Drug Target.    2009; 17(5):392-8. doi: 10.1080/10611860902902797. PubMed PMID:    WOS:000268243000005.-   8. Lux J, Vezeridis A M, Hoyt K, Adams S R, Armstrong A M, Sirsi S    R, Mattrey R F. Thrombin-Activatable Microbubbles as Potential    Ultrasound Contrast Agents for the Detection of Acute Thrombosis.    ACS Appl Mater Interfaces. 2017; 9(43):37587-96. Epub 2017/10/11.    doi: 10.1021/acsami.7b10592. PubMed PMID: 28994575; PMCID:    PMC5691601.-   9. Nakatsuka M A, Mattrey R F, Esener S C, Cha J N, Goodwin A P.    Aptamer-crosslinked microbubbles: smart contrast agents for    thrombin-activated ultrasound imaging. Adv Mater. 2012;    24(45):6010-6. Epub 2012/09/04. doi: 10.1002/adma.201201484. PubMed    PMID: 22941789; PMCID: PMC3626403.-   10. Mattrey R F, Steinbach G C. Ultrasound contrast agents. State of    the art. Invest Radiol. 1991; 26 Suppl 1:S5-11; discussion S5.    PubMed PMID: 1808149.-   11. Behan M, O'Connell D, Mattrey R F, Carney D N.    Perfluorooctylbromide as a contrast agent for C T and sonography:    preliminary clinical results. AJR Am J Roentgenol. 1993;    160(2):399-405. doi: 10.2214/ajr.160.2.8424361. PubMed PMID:    8424361.-   12. Kripfgans O D, Fowlkes J B, Miller D L, Eldevik O P, Carson P L.    Acoustic droplet vaporization for therapeutic and diagnostic    applications. Ultrasound Med Biol. 2000; 26(7):1177-89. PubMed PMID:    11053753.-   13. Schutt E G, Klein D H, Mattrey R M, Riess J G. Injectable    microbubbles as contrast agents for diagnostic ultrasound imaging:    the key role of perfluorochemicals. Angew Chem Int Ed Engl. 2003;    42(28):3218-35. doi: 10.1002/anie.200200550. PubMed PMID: 12876730.-   14. Beppu S, Matsuda H, Shishido T, Matsumura M, Miyatake K.    Prolonged myocardial contrast echocardiography via peripheral venous    administration of QW3600 injection (EchoGen): its efficacy and side    effects. J Am Soc Echocardiogr. 1997; 10(1):11-24. PubMed PMID:    9046489.-   15. Grayburn P A, Erickson J M, Escobar J, Womack L, Velasco C E.    Peripheral intravenous myocardial contrast echocardiography using a    2% dodecafluoropentane emulsion: identification of myocardial risk    area and infarct size in the canine model of ischemia. J Am Coll    Cardiol. 1995; 26(5):1340-7. doi: 10.1016/0735-1097(95)00306-1.    PubMed PMID: 7594052.-   16. Robbin M L, Eisenfeld A J. Perflenapent emulsion: a US contrast    agent for diagnostic radiology—multicenter, double-blind comparison    with a placebo. EchoGen Contrast Ultrasound Study Group. Radiology.    1998; 207(3):717-22. doi: 10.1148/radiology.207.3.9609895. PubMed    PMID: 9609895.-   17. Ho Y-J, Yeh C-K. Theranostic performance of acoustic nanodroplet    vaporization-generated bubbles in tumor intertissue. Theranostics.    2017; 7(6):1477-88. doi: 10.7150/thno.19099-   18. Chen C C, Sheeran P S, Wu S Y, Olumolade 00, Dayton P A,    Konofagou E E. Targeted drug delivery with focused    ultrasound-induced blood-brain barrier opening using    acoustically-activated nanodroplets. J Control Release. 2013;    172(3):795-804. doi: 10.1016/j.jconrel.2013.09.025. PubMed PMID:    24096019; PMCID: PMC3866692.-   19. Wu S Y, Fix S M, Arena C B, Chen C C, Zheng W, Olumolade 00,    Papadopoulou V, Novell A, Dayton P A, Konofagou E E. Focused    ultrasound-facilitated brain drug delivery using optimized    nanodroplets: vaporization efficiency dictates large molecular    delivery. Phys Med Biol. 2018; 63(3):035002. doi:    10.1088/1361-6560/aaa30d. PubMed PMID: 29260735; PMCID: PMC5823501.-   20. Moyer L C, Timbie K F, Sheeran P S, Price R J, Miller G W,    Dayton P A. High-intensity focused ultrasound ablation enhancement    in vivo via phase-shift nanodroplets compared to microbubbles. J    Ther Ultrasound. 2015; 3:7. doi: 10.1186/s40349-015-0029-4. PubMed    PMID: 26045964; PMCID: PMC4455327.-   21. Ferretti S, Allegrini P R, Becquet M M, McSheehy P M J. Tumor    Interstitial Fluid Pressure as an Early-Response Marker for    Anticancer Therapeutics. Neoplasia. 2009; 11(9):874-81. doi:    10.1593/neo.09554. PubMed PMID: WOS:000270038200006.-   22. Samuel S, Duprey A, Fabiilli M L, Bull J L, Fowlkes J B. In vivo    microscopy of targeted vessel occlusion employing acoustic droplet    vaporization. Microcirculation. 2012; 19(6):501-9. doi:    10.1111/j.1549-8719.2012.00176.x. PubMed PMID: 22404846; PMCID:    PMC3414215.-   23. Sheeran P S, Wong V P, Luois S, McFarland R J, Ross W D,    Feingold S, Matsunaga T O, Dayton P A. Decafluorobutane as a    phase-change contrast agent for low-energy extravascular ultrasonic    imaging. Ultrasound Med Biol. 2011; 37(9):1518-30. doi:    10.1016/j.ultrasmedbio.2011.05.021. PubMed PMID: 21775049; PMCID:    PMC4450864.-   24. Lo A H, Kripfgans O D, Carson P L, Rothman E D, Fowlkes J B.    Acoustic droplet vaporization threshold: effects of pulse duration    and contrast agent. IEEE Trans Ultrason Ferroelectr Freq Control.    2007; 54(5):933-46. PubMed PMID: 17523558.-   25. Hutter J C, Luu H M, Mehlhaff P M, Killam A L, Dittrich H C.    Physiologically based pharmacokinetic model for fluorocarbon    elimination after the administration of an octafluoropropane-albumin    microsphere sonographic contrast agent. J Ultrasound Med. 1999;    18(1):1-11. PubMed PMID: 9952073.-   26. Okada M, Albrecht T, Blomley M J, Heckemann R A, Cosgrove D O,    Wolf K J. Heterogeneous delayed enhancement of the liver after    ultrasound contrast agent injection—a normal variant. Ultrasound Med    Biol. 2002; 28(8):1089-92. PubMed PMID: 12217445.-   27. Caruso G, Martegani A, Aiani L, Borghi C, Verderame F, Campisi    A, Salvaggio G, Lagalla R, Cardinale A E. Heterogeneous delayed    enhancement of hepatic parenchyma after intravenous infusion of    sonographic contrast agent: a new hypothesis. Radiol Med. 2007;    112(1):56-63. doi: 10.1007/s11547-007-0120-1. PubMed PMID: 17310291.-   28. Al-Husein B, Abdalla M, Trepte M, Deremer D L, Somanath P R.    Antiangiogenic therapy for cancer: an update. Pharmacotherapy. 2012;    32(12):1095-111. doi: 10.1002/phar.1147. PubMed PMID: 23208836;    PMCID: PMC3555403.-   29. de Gracia Lux C, Vezeridis A M, Lux J, Armstrong A M, Sirsi S R,    Hoyt K, Mattrey R F. Novel method for the formation of monodisperse    superheated perfluorocarbon nanodroplets as activatable ultrasound    contrast agents. RSC Adv. 2017; 7(77):48561-8. doi:    10.1039/C7RA08971F. PubMed PMID: 29430294; PMCID: PMC5801773.-   30. Riess J G. Understanding the fundamentals of perfluorocarbons    and perfluorocarbon emulsions relevant to in vivo oxygen delivery.    Artif Cells Blood Substit Immobil Biotechnol. 2005; 33(1):47-63.    Epub 2005/03/17. PubMed PMID: 15768565.-   31. Miyoshi T, Kato S. Detailed Analysis of the Surface Area and    Elasticity in the Saturated    1,2-Diacylphosphatidylcholine/Cholesterol Binary Monolayer System.    Langmuir. 2015; 31(33):9086-96. Epub 2015/08/11. doi:    10.1021/acs.langmuir.5b01775. PubMed PMID: 26255826.-   32. Borden M A, Longo M L. Dissolution behavior of lipid    monolayer-coated, air-filled microbubbles: Effect of lipid    hydrophobic chain length. Langmuir. 2002; 18(24):9225-33. doi:    10.1021/1a026082h. PubMed PMID: WOS:000179428400018.-   33. Mountford P A, Thomas A N, Borden M A. Thermal activation of    superheated lipid-coated perfluorocarbon drops. Langmuir. 2015;    31(16):4627-34. Epub 2015/04/09. doi: 10.1021/acs.langmuir.5b00399.    PubMed PMID: 25853278.-   34. Kooiman K, Kokhuis TJA, van Rooij T, Skachkov I, Nigg A, Bosch J    G, van der Steen AFW, van Cappellen W A, de Jong N. DSPC or DPPC as    main shell component influences ligand distribution and binding area    of lipid-coated targeted microbubbles. Eur J Lipid Sci Tech. 2014;    116(9):1217-27. doi: 10.1002/ejlt.201300434. PubMed PMID:    WOS:000343000600014.-   35. Baskin J M, Prescher J A, Laughlin S T, Agard N J, Chang P V,    Miller I A, Lo A, Codelli J A, Bertozzi C R. Copper-free click    chemistry for dynamic in vivo imaging. P Natl Acad Sci USA. 2007;    104(43):16793-7. doi: DOI 10.1073/pnas.0707090104. PubMed PMID:    WOS:000250487600015.-   36. Chang P V, Prescher J A, Sletten E M, Baskin J M, Miller I A,    Agard N J, Lo A, Bertozzi C R. Copper-free click chemistry in living    animals. P Natl Acad Sci USA. 2010; 107(5):1821-6. doi:    10.1073/pnas.0911116107. PubMed PMID: WOS:000274296300006.-   37. Brudno Y, Desai R M, Kwee B J, Joshi N S, Aizenberg M, Mooney    D J. In Vivo Targeting through Click Chemistry. Chemmedchem. 2015;    10(4):617-20. doi: 10.1002/cmdc.201402527. PubMed PMID:    WOS:000351773300005.-   38. Whitehead S A, McNitt C D, Mattern-Schain S I, Carr A J, Alam S,    Popik V V, Best M D. Artificial Membrane Fusion Triggered by    Strain-Promoted Alkyne-Azide Cycloaddition. Bioconjugate Chemistry.    2017; 28(4):923-32. doi: 10.1021/acs.bioconjchem.6b00578.-   39. Righi M, Puleo G L, Tonazzini I, Giudetti G, Cecchini M,    Micera S. Peptide-based coatings for flexible implantable neural    interfaces. Sci Rep. 2018; 8(1):502. doi:    10.1038/s41598-017-17877-y. PubMed PMID: 29323135; PMCID:    PMC5765121.-   Simberg D, Mattrey R F. Targeting of perfluorocarbon microbubbles to    selective populations of circulating blood cells. Journal of drug    targeting; 2009, 17:392-8.-   PCT Publication number WO2009052057-   U.S. Pat. Publication number 20170176305-   U.S. Pat. Publication number 20160167061

1. A method of imaging a region of interest, the method comprising:providing bubbles to the region of interest, wherein the bubbles have anaverage bubble diameter between 200 nm and 10 μm; providing nanodropletsto the region of interest to increase the average diameter of thebubbles that are in the region of interest; and exposing the region ofinterest to an ultrasound stimulus to generate an image of the region ofinterest.
 2. The method of claim 1 wherein providing the nanodroplets tothe region of interest is performed subsequent to providing the bubblesto the region of interest.
 3. The method of claim 1 wherein the bubblesand the nanodroplets are provided to the region of interest in acomposition comprising the bubbles and the nanodroplets.
 4. The methodof claim 1 wherein the average diameter of the bubbles that are in theregion of interest is increased by an order of magnitude.
 5. The methodof claim 1 wherein the average diameter of the bubbles that are in theregion of interest is increased by up to two orders of magnitude.
 6. Themethod of claim 1 wherein the nanodroplets comprise a perfluorocarbon(PFC).
 7. A method of increasing bubble size, the method comprising:providing bubbles to a region of interest, wherein the bubbles have anaverage bubble diameter between 200 nm and 10 μm; providing nanodropletsproximal to the bubbles to increase the average diameter of the bubblesthat are in the region of interest.
 8. The method of claim 7 whereinproviding the nanodroplets proximal to the bubbles is performedsubsequent to providing the bubbles to the region of interest.
 9. Themethod of claim 7 wherein the bubbles and the nanodroplets are providedin a composition comprising the bubbles and the nanodroplets.
 10. Themethod of claim 7 wherein the average diameter of the bubbles that arein the region of interest is increased by an order of magnitude.
 11. Amethod of isolating targeted cell structures, the method comprising:providing bubbles to the targeted cell structures to couple the bubblesto the targeted cell structures and form complexes; providingnanodroplets to the bubbles to increase the average diameter of thebubbles and increase the buoyancy of the bubbles that are coupled to thetargeted cell structures; and isolating the complexes from non-targetedcell structures from within a sample.
 12. The method of claim 11 whereinisolating the complexes from the non-targeted cell structures within thesample comprises floating the complexes within the sample.
 13. Themethod of claim 11 wherein providing the nanodroplets to the bubbles isperformed subsequent to providing the bubbles to the targeted cellstructures.
 14. The method of claim 11 wherein the bubbles and thenanodroplets are introduced into the sample in a composition comprisingthe bubbles and the nanodroplets.
 15. The method of claim 11 wherein theaverage diameter of the bubbles is increased by at least an order ofmagnitude when the nanodroplets are provided to the bubbles.
 16. Themethod of claim 11 wherein the nanodroplets comprise a perfluorocarbon(PFC).
 17. A method of occluding a vessel, the method comprising:providing bubbles to the vessel, wherein the bubbles are targeted to thevessel such that the bubbles become coupled to the vessel; and providingnanodroplets to the vessel to increase the average diameter of thebubbles that are coupled to the vessel, wherein the size of the bubblesincreases until the vessel becomes occluded.
 18. The method of claim 17wherein the nanodroplets comprise a perfluorocarbon (PFC).
 19. Themethod of claim 17 wherein an average diameter of the microbubbles is atleast about 1 μm and an average diameter of the nanodroplets is nogreater than about 450 nm.
 20. The method of claim 17, wherein thevessel is a blood vessel within a tumor microvasculature of a subjectand providing the bubbles to the vessel comprises administering thebubbles to the subject; and wherein providing the nanodroplets to thevessel comprises administering the nanodroplets to the subject to treatthe tumor by inducing blockage of the tumor microvasculature viaexpansion of the bubbles.